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Background Long-lasting insecticidal nets (LLINs) are the primary malaria prevention and control intervention in many parts of sub-Saharan Africa. While LLINs are expected to last at least 3 years under normal use conditions, they can lose effectiveness because they fall out of use, are discarded, repurposed, physically damaged, or lose insecticidal activity. The contributions of these different interrelated factors to durability of nets and their protection against malaria have been unclear. Methods Starting in 2009, LLIN durability studies were conducted in seven countries in Africa over 5 years. WHO-recommended measures of attrition, LLIN use, insecticidal activity, and physical integrity were recorded for eight different net brands. These data were combined with analyses of experimental hut data on feeding inhibition and killing effects of LLINs on both susceptible and pyrethroid resistant malaria vectors to estimate the protection against malaria transmission—in terms of vectorial capacity (VC)—provided by each net cohort over time. Impact on VC was then compared in hypothetical scenarios where one durability outcome measure was set at the best possible level while keeping the others at the observed levels. Results There was more variability in decay of protection over time by country than by net brand for three measures of durability (ratios of variance components 4.6, 4.4, and 1.8 times for LLIN survival, use, and integrity, respectively). In some countries, LLIN attrition was slow, but use declined rapidly. Non-use of LLINs generally had more effect on LLIN impact on VC than did attrition, hole formation, or insecticide loss. Conclusions There is much more variation in LLIN durability among countries than among net brands. Low levels of use may have a larger impact on effectiveness than does variation in attrition or LLIN degradation. The estimated entomological effects of chemical decay are relatively small, with physical decay probably more important as a driver of attrition and non-use than as a direct cause of loss of effect. Efforts to maximize LLIN impact in operational settings should focus on increasing LLIN usage, including through improvements in LLIN physical integrity. Further research is needed to understand household decisions related to LLIN use, including the influence of net durability and the presence of other nets in the household.

Country-wide planning and coordination can improve sustainability of vectorborne disease control., Arthropod vectors transmit organisms that cause many emerging and reemerging diseases, and their control is reliant mainly on the use of chemical insecticides. Only a few classes of insecticides are available for public health use, and the increased spread of insecticide resistance is a major threat to sustainable disease control. The primary strategy for mitigating the detrimental effects of insecticide resistance is the development of an insecticide resistance management plan. However, few examples exist to show how to implement such plans programmatically. We describe the formulation and implementation of a resistance management plan for mosquito vectors of human disease in Zambia. We also discuss challenges, steps taken to address the challenges, and directions for the future.

In this semi-field study, a new polymer-enhanced deltamethrin formulation, K-Othrine® PolyZone, was compared to a standard deltamethrin product for residual activity against a susceptible strain of laboratory-reared Anopheles gambiae using standard WHO cone bioassays. Residual insecticide efficacy was recorded after exposure to metal, cement and wood panels maintained in experimental huts in sub-tropical environmental conditions in north central Florida, USA, and panels stored in a climate controlled chamber located at the Centers for Disease Control and Prevention, Georgia, USA. K-Othrine® PolyZone demonstrated 100% control on metal and cement panels 1 year post application and > 80% control on wood panels up to 6 mo. The new formulation should be considered for use in indoor residual spray programmes requiring long-term control of malaria vectors.

Background Indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) remain the cornerstones of malaria vector control. However, the development of insecticide resistance and its implications for operational failure of preventative strategies are of concern. The aim of this study was to characterize insecticide resistance among Anopheles arabiensis populations in Ethiopia and describe temporal and spatial patterns of resistance between 2012 and 2016. Methods Between 2012 and 2016, resistance status of An. arabiensis was assessed annually during the long rainy seasons in study sites from seven of the nine regions in Ethiopia. Insecticide resistance levels were measured with WHO susceptibility tests and CDC bottle bioassays using insecticides from four chemical classes (organochlorines, pyrethroids, organophosphates and carbamates), with minor variations in insecticides tested and assays conducted between years. In selected sites, CDC synergist assays were performed by pre-exposing mosquitoes to piperonyl butoxide (PBO). In 2015 and 2016, mosquitoes from DDT and deltamethrin bioassays were randomly selected, identified to species-level and screened for knockdown resistance (kdr) by PCR. Results Intense resistance to DDT and pyrethroids was pervasive across Ethiopia, consistent with historic use of DDT for IRS and concomitant increases in insecticide-treated net coverage over the last 15 years. Longitudinal resistance trends to malathion, bendiocarb, propoxur and pirimiphos-methyl corresponded to shifts in the national insecticide policy. By 2016, resistance to the latter two insecticides had emerged, with the potential to jeopardize future long-term effectiveness of vector control activities in these areas. Between 2015 and 2016, the West African (L1014F) kdr allele was detected in 74.1% (n = 686/926) of specimens, with frequencies ranging from 31 to 100% and 33 to 100% in survivors from DDT and deltamethrin bioassays, respectively. Restoration of mosquito susceptibility, following pre-exposure to PBO, along with a lack of association between kdr allele frequency and An. arabiensis mortality rate, both indicate metabolic and target-site mutation mechanisms are contributing to insecticide resistance. Conclusions Data generated by this study will strengthen the National Malaria Control Programme’s insecticide resistance management strategy to safeguard continued efficacy of IRS and other malaria control methods in Ethiopia. Electronic supplementary material The online version of this article (10.1186/s12936-017-2115-2) contains supplementary material, which is available to authorized users.

Background In this semi-field study, a new polymer-enhanced deltamethrin formulation, K-Othrine® PolyZone, was compared to a standard deltamethrin product for residual activity against a susceptible strain of laboratory-reared Anopheles gambiae using standard WHO cone bioassays. Methods Residual insecticide efficacy was recorded after exposure to metal, cement and wood panels maintained in experimental huts in sub-tropical environmental conditions in north central Florida, USA, and panels stored in a climate controlled chamber located at the Centers for Disease Control and Prevention, Georgia, USA. Conclusions K-Othrine® PolyZone demonstrated 100% control on metal and cement panels 1 year post application and > 80% control on wood panels up to 6 mo. The new formulation should be considered for use in indoor residual spray programmes requiring long-term control of malaria vectors. Electronic supplementary material The online version of this article (10.1186/s12936-018-2239-z) contains supplementary material, which is available to authorized users.

Surveillance of malaria vectors in Africa is most often accomplished using CDC-type light traps or human landing catches (HLCs). Over the past 30 yr, a variety of commercial and experimental mosquito traps have been developed for residential mosquito control or for improved surveillance of disease vector species, including the BG Sentinel (BGS) trap. To optimize collection of Anopheles gambiae Giles using this trap, BGS traps were modified with an opening (vent) added to the trap base to decrease exhaust airflow. Four traps configurations were tested with colony-reared host-seeking female An. gambiae in free-flying laboratory enclosures. Six attractant treatments (three attractants: BG-Lure, Limburger cheese, and a blank, with and without CO2) were tested concurrently. Across all trap-attractant combinations, significantly more mosquitoes (P < 0.05) were collected in standard, unmodified traps set upright (mean: 10.0) or upside down (9.8) than vented traps, whether upright (5.9) or upside down (7.0). The BG-Lure + CO2 and Limburger Cheese + CO2 bait combinations were more attractive than the other attractants tested alone. All attractant combinations collected significantly more mosquitoes than unbaited traps. Field studies are needed to determine if BG-Lure + CO2- or Limburger cheese + CO2-baited BGS traps are comparable with HLCs in collecting host-seeking An. gambiae.

Sprayer comparisons and larval morality assays were conducted following SR450 backpack mist blower and Superhawk XP thermal fogger applications of Vectobac® WDG Bacillus thuringiensis israelensis (Bti) against Culex quinquefasciatus. Bacillus thuringiensis israelensis was applied at maximum label rate in a 232.26-m2 field plot located in north-central Florida with containers placed at 2 heights (ground level and 1.52 m above ground) on stakes positioned 3.04, 6.09, 9.14, 12.19, and 15.24 m from the spray line. Results indicated that there was no significant (P > 0.05) difference in 24- and 48-h larval mortality between the 2 sprayers or between the 2 heights. There was significant difference (P 70% larval mortality 3.04–9.14 m from the spray line, and

Malaria vector mosquitoes belonging to the Anopheles gambiae complex were studied in four hamlets in The Gambia. All inhabitants were given bednets treated either with a placebo (milk) in two hamlets or with the pyrethroid insecticide permethrin (500 mg/m2) in two other hamlets. Malaria transmission occurred mainly during a few weeks of the rainy season, in September and October 1987. The indoor resting densities of mosquitoes in permethrin-treated hamlets were reduced, and we estimated over 90% reduction in biting on man by An. gambiae Giles sensu stricto in these hamlets. No mosquitoes were found under permethrin-treated bednets compared with eighty-one recovered from placebo-treated bednets. Mosquitoes exited more readily from rooms where permethrin-treated bednets were used than from rooms with placebo-treated nets. The annual mean probability that a child would receive an infective bite was estimated to be 0.09 in hamlets with insecticide-treated bednets, compared with 1.9 where placebo-treated bednets were used. Permethrin-treated bednets are therefore recommended as a means of effectively reducing the risk of exposure to malaria transmission, particularly in areas of low seasonal transmission.

The release of Wolbachia infected mosquitoes is likely to form a key component of disease control strategies in the near future. We investigated the potential of using near-infrared spectroscopy (NIRS) to simultaneously detect and identify two strains of Wolbachia pipientis (wMelPop and wMel) in male and female laboratory-reared Aedes aegypti mosquitoes. Our aim is to find faster, cheaper alternatives for monitoring those releases than the molecular diagnostic techniques that are currently in use. Our findings indicate that NIRS can differentiate females and males infected with wMelPop from uninfected wild type samples with an accuracy of 96% (N = 299) and 87.5% (N = 377), respectively. Similarly, females and males infected with wMel were differentiated from uninfected wild type samples with accuracies of 92% (N = 352) and 89% (N = 444). NIRS could differentiate wMelPop and wMel transinfected females with an accuracy of 96.6% (N = 442) and males with an accuracy of 84.5% (N = 443). This non-destructive technique is faster than the standard polymerase chain reaction diagnostic techniques. After the purchase of a NIRS spectrometer, the technique requires little sample processing and does not consume any reagents., Author Summary Near infrared spectroscopy (NIRS) is a technique that measures specific frequencies of light absorbed by C-H, O-H, S-H and N-H functional groups. Mosquito samples are grouped based upon absorption differences between their chemical properties. In this study, we used NIRS to differentiate 1) Aedes aegypti infected with either of the two strains of intracellular bacterium Wolbachia (wMel and wMelPop) from wild type Ae. aegypti and 2) Aedes aegypti infected with wMel from those infected with wMelPoP. NIRS facilitated the differentiation of wMel and wMelPop from wild type samples and samples infected with either of the Wolbachia infected strains with high prediction accuracies over their lifespan. Predictive models were derived from initial calibration data sets and validated against independent cohorts. Prediction accuracies were high (82–98%) regardless of the cohort mosquitoes were sampled from. The results show that NIRS may have real potential as an alternative method for monitoring Wolbachia incidence in mosquitoes. A rapid, simple and cost-effective surveillance tool suitable for resource-poor areas and large urban release programs would be of great utility for evaluating Wolbachia-based interventions. The models developed during this study require further validation using field collections.

Background With the emergence and spread of vector resistance to pyrethroids and DDT in Africa, several countries have recently switched or are considering switching to carbamates and/or organophosphates for indoor residual spraying (IRS). However, data collected on the residual life of bendiocarb used for IRS in some areas indicate shorter than expected bio-efficacy. This study evaluated the effect of pH and wall type on the residual life of the carbamates bendiocarb and propoxur as measured by the standard World Health Organization (WHO) cone bioassay test. Methods In phase I of this study, bendiocarb and propoxur were mixed with buffered low pH (pH 4.3) local water and non-buffered high pH (pH 8.0) local water and sprayed on two types of wall surface, mud and dung, in experimental huts. In the six month phase II study, the two insecticides were mixed with high pH local water and sprayed on four different surfaces: painted, dung, mud and mud pre-wetted with water. The residual bio-efficacy of the insecticides was assessed monthly using standard WHO cone bioassay tests. Results In phase I, bendiocarb mixed with high pH water killed more than 80 % of susceptible Anopheles arabiensis mosquitoes for two months on both dung and mud surfaces. On dung surfaces, the 80 % mortality threshold was achieved for three months when the bendiocarb was mixed with low pH water and four months when it was mixed with high pH water. Propoxur lasted longer than bendiocarb on dung surfaces, staying above the 80 % mortality threshold for four and five months when mixed with high and low pH water, respectively. Phase II results also showed that the type of surface sprayed has a significant impact on the bio-efficacy of bendiocarb. Keeping the spray water constant at the same high pH of 8.0, bendiocarb killed 100 % of exposed mosquitoes on impervious painted surfaces for the six months of the study period compared with less than one month on mud surfaces. Conclusions Mixing the insecticides in alkaline water did not reduce the residual bio-efficacy of bendiocarb. However, bendiocarb performed much better on impervious (painted) surfaces than on porous dung or mud ones. Propoxur was less affected by wall type than was bendiocarb. Studies on the interaction between wall materials, soil, humidity, temperature and pH and the residual bio-efficacy of new and existing insecticides are recommended prior to their wide use in IRS.

We used sentinel animal enclosures to measure the rate of infestation by the Chagas disease vector, Triatoma infestans, in an urban community of Arequipa, Peru, and to evaluate the effect of deltamethrin-impregnated netting on that rate. Impregnated netting decreased the rate of infestation of sentinel enclosures (rate ratio, 0.23; 95% confidence interval, 0.13-0.38; P < 0.001), controlling for the density of surrounding vector populations and the distance of these to the sentinel enclosures. Most migrant insects were early-stage nymphs, which are less likely to carry the parasitic agent of Chagas disease, Trypanosoma cruzi. Spread of the vector in the city therefore likely precedes spread of the parasite. Netting was particularly effective against adult insects and late-stage nymphs; taking into account population structure, netting decreased the reproductive value of migrant populations from 443.6 to 40.5. Impregnated netting can slow the spread of T. infestans and is a potentially valuable tool in the control of Chagas disease.

Long-term control of triatomine bugs in Chagas endemic regions will depend on a full understanding of vector-parasite-host interactions. Herein we describe a cytochrome b multiplex polymerase chain reaction (PCR)-based strategy for blood meal source identification in bug foregut contents. This technique discriminates human from animal blood, and has been tested in five Triatoma species from México. Host identification has been validated for human, four rodent species, two bat species, dog, rabbit, sheep, and opossum. In addition, Trypanosoma cruzi can be identified simultaneously using S34/S67-specific kinetoplast DNA primers. Both host and parasite identification were possible as long as 10 weeks after bug feeding, and in samples stored up to 6 years. The blood meal identification procedure described here represents a powerful tool for large-scale studies identifying the biological, ecological, and environmental variables associated with Chagas disease transmission.

Vector incrimination studies were conducted from April 2003 to February 2005 at three riverine villages 1.5 km to 7.0 km apart, along the Matapi River, Amapa State, Brazil. A total of 113,117 mosquitoes were collected and placed in pools of

Emerging and re-emerging vector-borne diseases such as chikungunya and dengue and associated Aedes vectors are expanding their historical ranges; thus, there is a need for the development of novel insecticides for use in vector control programs. The mosquito toxicity of a novel insecticide and repellent consisting of medium-chain carbon fatty acids (C8910) was examined. Determination of LC50 and LC90 was made against colony-reared Aedes aegypti (L.) and Aedes albopictus (Skuse) using probit analysis on mortality data generated by Centers for Disease Control and Prevention bottle bioassays. Six different concentrations of C8910 + silicone oil yielded an LC50 of 160.3 µg a.i/bottle (147.6–182.7) and LC90 of 282.8 (233.2–394.2) in Ae. aegypti ; five concentrations yielded an LC50 of 125.4 (116.1–137.6) and LC90 of 192.5 (165.0–278.9) in Ae. albopictus. Further development of C8910 and similar compounds could provide vector control specialists novel insecticides for controlling insect disease vectors.

Traditional sources of carbon dioxide (CO₂), dry ice, and compressed gas, were tested against 3 combinations of food-grade reagents known to generate CO₂using a compact, lightweight generator delivery system with Centers for Disease Control and Prevention light traps. Three 6 × 6 Latin square trials were completed near the Florida Gulf Coast in the Lower Suwannee Wildlife Refuge during the summer of 2013, collecting a total of 31,632 female mosquitoes. Treatments included dry ice, compressed CO₂gas, a control trap (no CO₂), citric acid + sodium bicarbonate, vinegar + sodium bicarbonate, and yeast + sugar. Decreasing order of trap collections (treatment mean number of mosquitoes per trap night ± standard error) were dry ice 773.5 (± 110.1) > compressed gas 440.7 (± 42.3) > citric acid + sodium bicarbonate 197.6 (± 30.4), yeast + sugar 153.6 (± 27.4) > vinegar + sodium bicarbonate 109.6 (± 16.2) > control 82.4 (± 14.0). A 2-way Kruskal-Wallis analysis by treatment, site, and treatment × site interaction identified significant differences between all treatments. Although dry ice and compressed CO₂gas collected significantly more mosquitoes than other combinations (P < 0.05), use of citric acid and sodium bicarbonate or yeast and sugar greatly outperformed unbaited traps and offer a good alternative to dry ice and compressed gas in areas where these agents are not readily available or are difficult to obtain due to logistical constraints. An inexpensive, portable CO₂generator for use with food-grade reagents is described.

Background Long-lasting insecticidal nets (LLINs) are a mainstay of malaria prevention in Africa. More LLINs are available now than in any time previously due to increases in funding for malaria control. LLINs are expected to last three to five years before they need to be replaced. Reports of nets lasting less than three years are frequent in Zambia, which, if true, will increase the number of LLINs needed to maintain universal coverage. Methods This study collected nets distributed during mass distribution campaigns. One net was collected from each participating home in 12 districts in 2010 and all nets were examined for holes. One household member was surveyed about net use and care. Results The study collected 713 polyester nets with a median age of 31 months (range 27–44 months, interquartile (IQR) range: 29–36 months), median number of holes was 17 (IQR: 5–33), and median total hole size was 88.3 sq cm (IQR: 14.5-360.4). The median total number of holes did differ by age of the net, from 27–44 months, but not in a linear fashion. The difference in the number of holes in the newest and oldest nets was not statistically significant. The mean deltamethrin level for all nets was 23 mg/sq m (≥8 mg/sq m is considered effective). There was a larger total hole area in the lower half of the nets (repeat measures ANOVA, F = 228.43, df = 2, p

Interventions targeting adult mosquitoes are used to combat transmission of vector-borne diseases, including dengue. Without available vaccines, targeting the primary vector, Aedes aegypti, is essential to prevent transmission. Older mosquitoes (≥ 7 days) are of greatest epidemiological significance due to the 7-day extrinsic incubation period of the virus. Age-grading of female mosquitoes is necessary to identify post-intervention changes in mosquito population age structure. We developed models using near-infrared spectroscopy (NIRS) to age-grade adult female Ae. aegypti. To determine if diet affects the ability of NIRS models to predict age, two identical larval groups were fed either fish food or infant cereal. Adult females were separated and fed sugar water ± blood, resulting in four experimental groups. Females were killed 1, 4, 7, 10, 13, or 16 days postemergence. The head/thorax of each mosquito was scanned using a near-infrared spectrometer. Scans from each group were analyzed, and multiple models were developed using partial least squares regression. The best model included all experimental groups, and positively predicted the age group (< or ≥ 7 days) of 90.2% mosquitoes. These results suggest both larval and adult diets can affect the ability of NIRS models to accurately assign age categories to female Ae. aegypti.

Sprayer comparisons and larval morality assays were conducted following SR450 backpack mist blower and Superhawk XP thermal fogger applications of Vectobac® WDG Bacillus thuringiensis israelensis (Bti) against Culex quinquefasciatus. Bacillus thuringiensis israelensis was applied at maximum label rate in a 232.26-m(2) field plot located in north-central Florida with containers placed at 2 heights (ground level and 1.52 m above ground) on stakes positioned 3.04, 6.09, 9.14, 12.19, and 15.24 m from the spray line. Results indicated that there was no significant (P0.05) difference in 24- and 48-h larval mortality between the 2 sprayers or between the 2 heights. There was significant difference (P0.05) among the 5 rows, with mortality continuously decreasing with increasing distance from sprayer. Both sprayers provided on average70% larval mortality 3.04-9.14 m from the spray line, and60% mortality at 12.19 and 15.24 m. The data suggest that the SR450 and Superhawk XP may be comparable sprayers for use with Bti to control mosquito larvae.

We present evidence that a parasite with characteristics of Plasmodium vivax is being transmitted among Duffy blood group-negative inhabitants of Kenya. Thirty-two of 4,901 Anopheles gambiae and An. funestus (0.65%) collected in Nyanza Province were ELISA positive for the P. vivax circumsporozoite protein VK 247. All positives were found late in the rainy season, when An. funestus predominated, and disproportionately many were found at a single village. A P. vivax specific sequence of the SSU rRNA gene was amplified from three of six ELISA-positive mosquitoes. Erythrocytes from 31 children, including 9 microscopically diagnosed as infected with P. vivax, were negative by flow cytometry for the Fy3 or Fy6 epitopes, which indicate Duffy blood group expression. A DNA fragment specific for the C terminus of the gene for P. vivax merozoite surface protein 1 (MSP-1) was amplified from the blood of four of these children and subsequently sequenced from two.

Hosts of blood-fed anophelines (Diptera: Culicidae) were determined in three riverine villages, 1.5-7.0 km apart, along the Matapi River, Amapa state, Brazil, by enzyme-linked immunosorbent assay midgut analysis for IgG of common vertebrates. Anopheles marajoara Galvao & Damsceno and Anopheles darlingi Root had higher human blood indices (HBI) than Anopheles nuneztovari Gabaldon, Anopheles triannulatus (Neiva and Pinto), and Anopheles intermedius (Chagas), which were relatively zoophilic. HBIs of An. darlingi varied significantly among villages, attributable to a low proportion of human-fed mosquitoes in Santo Antonio. Significantly higher incidence of An. marajoara and An. nuneztovari fed on pig blood at two villages, associated with a low number of pigs in Santo Antonio. The incidences of bovine blood varied significantly among villages for all three of the most common anopheline species. The incidence of mixed meals ranged from 7.1 to 27.6% among common species, and, for An. marajoara, varied significantly among villages. This study demonstrates differences in host selection patterns among villages only a few kilometers apart, which may be influenced by host availability and have important epidemiological consequences.

In several districts of Boa Vista, state of Roraima, Brazil we found Anopheles (Nyssorhynchus) albitarsis E to be the primary vector of human malaria parasites, and during 2001-2002 it was significantly more abundant than An. darlingi (p < 0.001). Other species sampled were An. (Nys.) braziliensis, An. (Ano.) peryassui, An. (Nys.) nuneztovari, An. (Nys.) oswaldoi s.l., and An. (Nys.) triannulatus. As determined by the ELISA technique An. darlingi had a higher overall infection rate (2.1%) compared with An. albitarsis E (1.2%). However, a marginally higher proportion of An. albitarsis E was infected with Plasmodium vivax compared with An. darlingi, and the An. albitarsis E biting index was also much higher. These results suggest the importance of An. albitarsis E in malaria transmission in a savannah ecoregion of northern Amazonian Brazil, and reconfirm the importance of An. darlingi even if at lower abundance.

Implementation of the sterile insect technique for tsetse (Glossina spp.) requires that only sterile male insects be released; thus, at some stage of the fly production process the females have to be removed. A further constraint in the use of the sterile insect technique for tsetse is that the females are needed for colony production and hence, a non-destructive method of sex separation is required. In most tsetse sterile insect technique programmes thus far, females have been eliminated from the released material by hand-separation of chilled adults. Using near-infrared (NIR) spectroscopy, significant differences have been found between the spectra for the pupae of male and female G. pallidipes Austen. Significantly, the differences appear to be maximized 4-5 days before emergence of the adults. Tsetse fly pupae up to five days before emergence can be sexed with accuracies that generally range from 80 to 100%. This system, when refined, will enable effective separation of male and female pupae to be carried out, with emerged females being returned to the colony and males being irradiated and released. If separation can be achieved five days before emergence, this will also enable irradiated male pupae to be shipped to other destinations as required. Other Diptera were evaluated using this system but had lower classification accuracies of 50-74%. This may be due to the difference in reproductive physiology between these different fly groups.

Two recent outbreaks of locally acquired, mosquito-transmitted malaria in Virginia in 1998 and 2002 demonstrate the continued risk of endemic mosquito-transmitted malaria in heavily populated areas of the eastern United States. Increasing immigration, growth in global travel, and the presence of competent anopheline vectors throughout the eastern United States contribute to the increasing risk of malaria importation and transmission. On August 23 and 25, 2002, Plasmodium vivax malaria was diagnosed in 2 teenagers in Loudoun County, Virginia. The Centers for Disease Control and Prevention (CDC) deemed these cases to be locally acquired because of the lack of risk factors for malaria, such as international travel, blood transfusion, organ transplantation, or needle sharing. The patients lived approximately 0.5 mi apart; however, 1 patient reported numerous visits to friends who lived directly across the street from the other patient. Two Anopheles quadrimaculatus s.l. female pools collected in Loudoun County, Virginia, and 1 An. punctipennis female pool collected in Fairfax County, Virginia, tested positive for P. vivax 210 with the VecTest panel assay and enzyme-linked immunosorbent assay (ELISA). In addition, 2 An. quadrimaculatus s.l. female pools collected in Montgomery, Maryland, tested positive for P. vivax 210. The CDC confirmed these initial results with the circumsporozoite ELISA. The authors believe that this is the 1st demonstration of Plasmodium-infected mosquitoes collected in association with locally acquired human malaria in the United States since the current national malaria surveillance system began in 1957.

We evaluated the performance of the VecTest Malaria Antigen Panel (V-MAP) assay for the detection of Plasmodium falciparum and P. vivax (variants 210 and 247) circumsporozoite protein in anopheline mosquitoes in Thailand. The V-MAP assay is a rapid, one-step procedure using a 'dipstick' wicking test strip. The circumsporozoite (CS) ELISA was used as the reference standard. Mosquitoes evaluated in the study included field-collected specimens (n = 930) and laboratory-reared specimens that had been fed on blood collected from patients with and without Plasmodium gametocytes (n = 4,110) or on cultured P. falciparum gametocytes (n = 262). Field-collected mosquitoes were triturated individually or in pools of 2-5 and tested using 613 V-MAP assays. Laboratory-reared specimens were tested individually using 4,372 V-MAP assays. Assay performance depended on the species of Plasmodium and the number of sporozoites used as the cut-off. For P. falciparum, optimal performance was achieved using a cut-off of 150 sporozoites (sensitivity = 100%, specificity = 99.2%, and accuracy = 0.99). For P. vivax variant 210, optimal performance was also achieved using a cut-off of 150 sporozoites (sensitivity = 94.8%, specificity = 94.5%, and accuracy = 0.95). We were unable to develop a standard-curve for the CS-ELISA using P. vivax variant 247 because of a lack of sporozoites; however, using a cut-off of 30 pg P. vivax 247 antigen (mosquitoes with less than this amount of antigen were considered negative), assay performance (sensitivity = 94.3%, specificity = 99.2%, and accuracy = 0.99) was comparable to that achieved for P. falciparum and P. vivax 210. These results clearly demonstrate that the V-MAP assay performs at an acceptable level and offers practical advantages for field workers needing to make rapid surveys of malaria vectors.

An evaluation of malaria transmission and epidemiology in the Amazonian city of Belém over the last 70 years shows that (1) Anopheles darlingi, reported to be eradicated in 1968, reappeared in the mid 1990s, with a marked increase in abundance between 1997 to 1999 in two of three districts sampled; (2) An. darlingi and An. aquasalis are each implicated in current malaria transmission in different districts of the city; (3) mosquito species diversity (in Anopheles subgenus Nyssorhynchus) has increased from two in the 1930s to six in the 1940s to 10 in the 1990s; (4) there is no overall correlation between malaria case incidence and human population size from 1940 to 1996 in Belém; (5) however, the total number of malaria cases has increased significantly since the late 1970s and over the short term from 1993 to 1999; and (6) interestingly, the short term increases are due solely to cases of Plasmodium vivax infection; cases of P. falciparum malaria are declining (significantly for Pará state only). The reappearance of An. darlingi may be a result of the continued expansion of Belém into the surrounding forest in the 1990s. In the absence of preventative measures, we predict an increase in local outbreaks of malaria in the DAENT and DAICO districts where the population sizes of An. darlingi are increasing.

The recent outbreaks of West Nile (WN) encephalitis and St. Louis encephalitis (SLE) in the United States have highlighted the need for rapid and specific methods of detecting arboviral antigens in mosquitoes. We evaluated rapid, field-usable assays for detecting and differentiating WN and SLE viruses in mosquito pools, based on a patent-pending, immunochromatographic technology (VecTest) formatted on a dipstick. The device provides results in less than 20 min and can be used in laboratories with adequate containment facilities. In laboratory assessments, both the SLE and WN virus tests demonstrated sensitivity comparable with that of an antigen capture ELISA, but less than can be achieved with Vero cell plaque or reverse-transcriptase polymerase chain reaction assays. There was no evidence of cross-reaction when tested with high concentrations of heterologous flavivirus antigens or with Eastern equine encephalitis or Western equine encephalitis viruses. Both the WN and SLE dipstick tests delivered a clear positive result with a single positive specimen in a pool of 50 mosquitoes. This virus assay technology reduces the time required to obtain test results and will allow rapid medical threat assessment and effective targeting of vector control measures.

To determine which species and populations of Anopheles transmit malaria in any given situation, immunological assays for malaria sporozoite antigen can replace traditional microscopical examination of freshly dissected Anopheles. We developed a wicking assay for use with mosquitoes that identifies the presence or absence of specific peptide epitopes of circumsporozoite (CS) protein of Plasmodium falciparum and two strains of Plasmodium vivax (variants 210 and 247). The resulting assay (VecTest Malaria) is a rapid, one-step procedure using a 'dipstick' test strip capable of detecting and distinguishing between P. falciparum and P. vivax infections in mosquitoes. The objective of the present study was to test the efficacy, sensitivity, stability and field-user acceptability of this wicking dipstick assay. In collaboration with 16 test centres world-wide, we evaluated more than 40 000 units of this assay, comparing it to the standard CS ELISA. The 'VecTest Malaria' was found to show 92% sensitivity and 98.1% specificity, with 97.8% accuracy overall. In accelerated storage tests, the dipsticks remained stable for > 15 weeks in dry conditions up to 45 degrees C and in humid conditions up to 37 degrees C. Evidently, this quick and easy dipstick test performs at an acceptable level of reliability and offers practical advantages for field workers needing to make rapid surveys of malaria vectors.

To figure out how far spread the malaria parasite infected mosquitoes from Demilitarized Zone (DMZ). We investigated the sporozoite-infected mosquitoes in Chosan-ri and Hajiseok-ri of Paju-shi, Tonae-dong of Koyang-shi, Mudeung-ri of Yonchon-gun, Soyo-dong of Tongduchon-shi, and Tukjeong-ri of Yangju-gun, Kyonggi-do province in 1998. They were known as highly epidemic areas in the Republic of Korea. Anopheline mosquitoes were collected by human biting collection method from the 2nd week of June to the 5th week of August. A total of 3,256 anopheles mosquitoes were captured in these areas. A sandwich enzyme-linked immununosorbent assay (ELISA) for identifying Plasmodium vivax sporozoites in mosquitoes was used in this survey. Only one mosquito which was captured in Chosan-ri, on 2nd week of July showed positive reaction in this test. This mosquito was identified as Anopheles sinensis. This showed a 0.068% (1/1,465) positive rate in Chosan-ri on 2nd week of July. We could not detect positive mosquitoes the other areas. So we concluded the parasite infected mosquitoes may be highly limited near the DMZ.

Phylogenetic relationships among thirty-two species of mosquitoes in subfamily Anophelinae are inferred from portions of the mitochondrial genes COl and COII, the nuclear 18S small subunit rRNA gene and the expansion D2 region of the nuclear large subunit 28S rRNA gene. Sequences were obtained from the genera Anopheles, Bironella and Chagasia. Representatives of all six sub- genera of Anopheles were included: Anopheles, Cellia, Kerteszia, Lophopodomyia, Nyssorhynchus and Stethomyia. Using parsimony and maximum likelihood meth- ods, various combinations of these DNA sequence data were analysed separately: 18S, 28S, combined 18S and 28S, combined CO1 and COIl, and combined 18S, 28S, COl and COII ('total evidence'). The combined rDNA data contain strong phylogenetic signal, moderately to strongly supporting most clades in MP and ML analyses; however, the mtDNA data (analysed as either nucleotide or amino acid sequences) contain little phylogenetic signal, except for relationships of very recently derived groups of species and, at the deepest level, for the monophyly of Anophelinae. The paraphyly of Anopheles relative to Bironella is confirmed by most analyses and statistical tests. Support for the monophyly of subgenera Anopheles, Cellia, Kerteszia and N)'ssorhynchus is indicated by most analyses. Subgenus Lophopodomyia is reconstructed as the sister to Bironella, nested within a clade also containing Nyssorhynchus and Kerteszia. The most basal relationships within genus Anopheles are not well resolved by any of the data partitions, although the results of statistical analyses of the rDNA data (S-H-tests, likelihood ratio tests for monophyly and Bayesian MCMC analyses) suggest that the clade consisting of Bironella, Lophopodom)'ia, Nyssorhynchus and Kerteszia is the sister to the clade containing Cellia and Anopheles.

The sensitivity and accuracy of near-infrared spectroscopy (NIRS) was compared with that of the pteridine fluorescence technique for estimating the chronological age of house flies, Musca domestica (L.). Although results with both techniques were significantly correlated with fly age, confidence limits on predicted ages generally were smaller with NIRS. Young flies could be readily differentiated from old flies by using NIRS. Age predictions using the pteridine method are dependent upon size, sex, and temperature at which adult flies are exposed. In contrast, those factors do not need to be determined for age-grading using NIRS. Classification accuracy using the NIRS method was similar for whole flies, fresh heads, dried heads, and ethanol-preserved heads. The NIRS method was also suitable for predicting age of stable flies, Stomoxys calcitrans (L.), and face flies, Musca autumnalis De Geer. NIRS has several advantages over the measurement of pteridine levels for age-grading field-collected flies, including speed and portability of instrumentation, and not needing to determine sex, size, and temperatures to which adult flies were exposed.

Surveillance of malaria vectors in Africa is most often accomplished using CDC-type light traps or human landing catches (HLCs). Over the past 30 yr, a variety of commercial and experimental mosquito traps have been developed for residential mosquito control or for improved surveillance of disease vector species, including the BG Sentinel (BGS) trap. To optimize collection of Anopheles gambiae Giles using this trap, BGS traps were modified with an opening (vent) added to the trap base to decrease exhaust airflow. Four traps configurations were tested with colony-reared host-seeking female An. gambiae in free-flying laboratory enclosures. Six attractant treatments (three attractants: BG-Lure, Limburger cheese, and a blank, with and without CO2) were tested concurrently. Across all trap-attractant combinations, significantly more mosquitoes (P0.05) were collected in standard, unmodified traps set upright (mean: 10.0) or upside down (9.8) than vented traps, whether upright (5.9) or upside down (7.0). The BG-Lure + CO2 and Limburger Cheese + CO2 bait combinations were more attractive than the other attractants tested alone. All attractant combinations collected significantly more mosquitoes than unbaited traps. Field studies are needed to determine if BG-Lure + CO2- or Limburger cheese + CO2-baited BGS traps are comparable with HLCs in collecting host-seeking An. gambiae.

Malaria remains the most serious vector-borne disease, affecting some 300-500 million people annually, transmitted by many species of Anopheles mosquitoes (Diptera: Culicidae). Monoclonal antibodies developed against specific circumsporozoite (CS) proteins of the main malaria parasites Plasmodium falciparum and P. vivax have been used previously for enzyme-linked immunosorbent assays (ELISA), widely employed for detection of malaria sporozoites in vector Anopheles for local risk assessment, epidemiological studies and targeting vector control. However, ELISA procedures are relatively slow and impractical for field use. To circumvent this, we developed rapid wicking assays that identify the presence or absence of specific peptide epitopes of CS protein of the most important P. falciparum and two strains (variants 210 and 247) of the more widespread P. vivax. The resulting assay is a rapid, one-step procedure using a 'dipstick' wicking test strip. In laboratory assessment, dipsticks identified 1 ng/ mL of any of these three CS protein antigens, with sensitivity nearly equal to the CS standard ELISA. We have developed and are evaluating a combined panel assay that will be both qualitative and quantitative. This quick and easy dipstick test (VecTest Malaria) offers practical advantages for field workers needing to make rapid surveys of malaria vectors.

The mosquito midgut plays a central role in the sporogonic development of malaria parasites. We have found that polyclonal sera, produced against mosquito midguts, blocked the passage of Plasmodium falciparum ookinetes across the midgut, leading to a significant reduction of infections in mosquitoes. Anti-midgut mAbs were produced that display broad-spectrum activity, blocking parasite development of both P. falciparum and Plasmodium vivax parasites in five different species of mosquitoes. In addition to their parasite transmission-blocking activity, these mAbs also reduced mosquito survivorship and fecundity. These results reveal that mosquito midgut-based antibodies have the potential to reduce malaria transmission in a synergistic manner by lowering both vector competence, through transmission-blocking effects on parasite development, and vector abundance, by decreasing mosquito survivorship and egg laying capacity. Because the intervention can block transmission of different malaria parasite species in various species of mosquitoes, vaccines against such midgut receptors may block malaria transmission worldwide.

We examined geographically distinct isolates of Plasmodium vivax and categorized them according to developmental success in Anopheles albimanus. We found that parasites from Central America and Colombia form a group distinct from those of Asia. New World isolates have a distinct chromosomal translocation and an episomal variation in the open reading frame (ORF) 470 DNA sequence that distinguishes them from the other isolates tested. Old World types of P. vivax were introduced into the Americas, and a remnant of this lineage remains in P. simium. It is indistinguishable from Old World P. vivax to the extent determinable by using our encoded markers and the examination of its developmental pattern in mosquitoes. The cohesive characteristics that separate types of P. vivax are predictors of range and potential for transmission and hence require taxonomic distinction.

A clinical trial (n = 120, 60 males and 60 females) was conducted to assess the efficacy of an extended duration tropical insect/arthropod repellent (EDTIAR) topical formulation of N,N-diethyl-m-toluamide (DEET). The amount of EDTIAR (mean +/- confidence interval), applied by participants in accordance with label directions, was not significantly different between females (3.66 +/- 0.32 mg/cm2) and males (3.45 +/- 0.33 mg/cm2). There also was no significant difference in the number of Anopheles stephensi mosquitoes biting the control arm of females or males at 0, 3, 6, 9, and 12 hr. While gender had no effect on feeding, the time of day did effect mosquito feeding with fewer mosquitoes feeding in the afternoon than in the morning or evening. The percent protective efficacy at 0, 3, 6, 9, and 12 hr was 100.0, 99.3, 92.8, 79.7 and 66.3 for females, and 100.0, 100.0, 97.6, 91.9, and 77.5 for males. These data are inconsistent with the EDTIAR label claim that the repellent provides 95% or greater protection against mosquitoes for 12 hr or more under normal use conditions. The results of a multivariate regression analysis indicated that 1) protection decreased linearly as time after application of repellent increased (P < 0.001), 2) individuals who applied higher doses of repellent were better protected against mosquito bites (P < 0.001), 3) females experienced significantly less protection over time than did males (P = 0.005), and 4) the estradiol concentration in the blood had no effect on efficacy of the repellent (P = 0.110).

Gonzalez-Ceron, L., Rodriguez, M. H., Wirtz, R. A., Sina, B. J., Palomeque, O. L., Nettel, J. A., and Tsutsumi, V. 1998.Plasmodium vivax:A monoclonal antibody recognizes a circumsporozoite protein precursor on the sporozoite surface.Experimental Parasitology90, 203–211. The major surface circumsporozoite (CS) proteins are known to play a role in malaria sporozoite development and invasion of invertebrate and vertebrate host cells.Plasmodium vivaxCS protein processing during mosquito midgut oocyst and salivary gland sporozoite development was studied using monoclonal antibodies which recognize different CS protein epitopes. Monoclonal antibodies which react with the CS amino acid repeat sequences by ELISA recognized a 50-kDa precursor protein in immature oocyst and additional 47- and 42-kDa proteins in older oocysts. A 42-kDa CS protein was detected after initial sporozoite invasion of mosquito salivary glands and an additional 50-kDa precursor CS protein observed later in infected salivary glands. These data confirm previous results with otherPlasmodiumspecies, in which more CS protein precursors were detected in oocysts than in salivary gland sporozoites. A monoclonal antibody (PvPCS) was characterized which reacts with an epitope found only in the 50-kDa precursor CS protein. PvPCS reacted with allP. vivaxsporozoite strains tested by indirect immunofluorescent assay, homogeneously staining the sporozoite periphery with much lower intensity than that produced by anti-CS repeat antibodies. Immunoelectron microscopy using PvPCS showed that the CS protein precursor was associated with peripheral cytoplasmic vacuoles and membranes of sporoblast and budding sporozoites in development oocysts. In salivary gland sporozoites, the CS protein precursor was primarily associated with micronemes and sporozoite membranes. Our results suggest that the 50-kDa CS protein precursor is synthesized intracellularly and secreted on the membrane surface, where it is proteolytically processed to form the 42-kDa mature CS protein. These data indicate that differences in CS protein processing in oocyst and salivary gland sporozoites development may occur.

The malaria sporozoite vaccine candidate RTS,S, formulated with an oil-in-water emulsion plus the immunostimulants monophosphoryl lipid A and the saponin derivative QS21 (vaccine 3), recently showed superior efficacy over two other experimental formulations. Immunized volunteers were followed to determine the duration of protective immune responses. Antibody levels decreased to between one-third and one-half of peak values 6 months after the last dose of vaccine. T cell proliferation and interferon-gamma production in vitro were observed in response to RTS,S or hepatitis B surface antigen. Seven previously protected volunteers received sporozoite challenge, and 2 remained protected (1/1 for vaccine 1, 0/1 for vaccine 2, and 1/5 for vaccine 3). The prepatent period was 10.8 days for the control group and 13.2 days for the vaccinees (P < .01). Immune responses did not correlate with protection. Further optimization in vaccine composition and/or immunization schedule will be required to induce longer-lasting protective immunity.

Summary The prokaryotic, spirochaetal microorganism Borrelia burgdorferi is the causative agent of Lyme disease, an arthropod-borne disease of a variety of vertebrates and the most prevalent arthropod-borne disease of humans in the United States. In order to understand better the normal life cycle of B. burgdorferi, an experimental chain of infection was devised that involved multiple sequential arthropod and mammalian passages. By examining populations of B. burgdorferi emerging from different points in this infectious chain, we demonstrate that selection of B. burgdorferi populations peculiar to arthropod or vertebrate hosts is a property of at least one of the two ecologically distinct strains we examined. Distinct B. burgdorferi populations were identified using an antigenic profile, defined by a set of monoclonal antibodies to eight B. burgdorferi antigens, and a plasmid profile, defined by the naturally occurring plasmids in the starting clonal populations. These two profiles constituted the phenotypical signature of the population. In the strain exhibiting selection in the different hosts, transition from one host to another produced a striking series of alternating phenotypical signatures down the chain of infection. At the molecular level, the alternating signatures were manifested as a reciprocal relationship between the expression of certain antigenic forms of outer surface protein (Osp) B and OspC. In the case of OspC, the antigenic changes could be correlated to the presence of one of two distinctly different alleles of the ospC gene in a full-length and presumably transcriptionally active state. In the case of OspB, two alleles were again identified. However, their differences were minor and their relationship to OspB antigenic variation more complicated. In addition to the reciprocating changes in the antigenic profile, a reciprocating change in the size (probably the multimeric state) of a 9.0 kbp supercoiled plasmid was also noted. Selection of distinct populations in the tick may be responsible for the microorganism’s ability to infect a wide range of vertebrate hosts efficiently, in that the tick might provide selective pressure for the elimination of the population selected in the previous host.

This study examined U.S. Army soldiers' knowledge, attitudes, and practices regarding personal protection measures (PPMs) to prevent arthropod-related diseases and nuisance bites. Soldiers attending 1 of 13 U.S. Army training courses either completed a written questionnaire (N = 1,007) or participated in a group interview (N = 65). Respondents primarily used repellents to prevent nuisance insect bites (55%) rather than to prevent disease (38.7%). Less than one-third correctly identified the military-issue repellents to be used on skin (31.7%) or clothing (26.1%). More than half (57.6%) thought that commercial products were better than military-issue repellents, but most (74.2%) reported that they did not have enough or any information about the U.S. military's system of PPMs. Soldiers have poor knowledge of U.S. military doctrine regarding PPMs and still prefer to use commercial products. A focused strategy to ensure the appropriate use of PPMs by service members should be developed, implemented, and evaluated.

Collections of human sera from malaria-endemic areas would be valuable for identifying and characterizing antigens as malaria vaccine candidates if the contributing serum donors' ability to resist infection were fully characterized. We prepared such a serum collection from 26 apparently immune Nigerian adults who failed to develop patent parasitemia for at least 20 weeks following a documented increase in antibodies to the circumsporozoite protein (CSP) from Plasmodium falciparum. Volunteers were evaluated five times per week for malaria symptoms and bimonthly for parasites by examining thick blood smears. The incidence rate over 13 months for the cohort was 42% (47 malaria-confirmed volunteers) and the risk of infection was 1.3 infections/year. Responses to CSP did not correlate with protection. Because antibody responses to antigens other than CSP may be associated with protection, the sera from these immune individuals may be useful for identifying and characterizing other potential malaria vaccine candidates.

We have analyzed RNA isolated from recently prepared and historically preserved slides containing smears of Plasmodium-infected blood. We found that slides preserved as long as 20 years can yield RNA that is a suitable template for polymerase chain reaction (PCR) amplification. Mosquitoes that have been stored for years under ambient temperature can also be used as an RNA source. The RNA amplification from slide-derived material is shown to be dependent upon the addition of reverse transcriptase and the resultant products are specific to the developmental state of the parasite. Amplification of ribosomal RNA with primers conserved for Plasmodium and hybridization with species-specific probes provide a general, unbiased method for species determination. Messenger RNA transcripts from slides also appear to serve as templates. The procedure may add complementary information to that derived from microscopic examination of Giemsa-stained blood smears including species identification, variant antigen identification and drug resistance status.

We have isolated a small, heat stabile, hydrophilic molecule from the gut lumen of unfed, female Anopheles stephensi that is a potent inducer of gametogenesis in Plasmodium falciparum and P. gallinaceum at a hydrogen ion concentration, pH 7.4, that normally suppresses activation. This gamete activation factor (GAF) was purified using reverse phase high performance liquid chromatography and determined to have a major ion m/z of 206.1 by low resolution electrospray mass spectrometry. The molecule, which was also found in the heads of both female and male A. stephensi, absorbed light in the ultraviolet region at three maxima (lambda(max) = 213, 245 and 350 nm); the 245/350 nm absorbance ratio was 7.0. The structure of the molecule and its normal function in the mosquito are not yet known, but in a sample of diverse insect species, extracts from those that feed on blood were bioactive. We propose that GAF is the previously observed malaria exflagellation factor (MEF).

The human malaria parasite Plasmodium vivax has been shown to regulate the transcription of two distinct 18 RNAs during development. Here we show a third and distinctive type of ribosome that is present shortly after zygote formation, a transcriptional pattern of ribosome types that relates closely to the developmental state of the parasite and a phenomenon that separates ribosomal types at a critical phase of maturation. The A-type ribosome is predominantly found in infected erythrocytes of the vertebrate and the mosquito blood meal. Transcripts from the A gene are replaced by transcripts from another locus, the O gene, shortly after fertilization and increase in number as the parasite develops on the mosquito midgut. Transcripts from another locus, the S gene, begins as the oocyst form of the parasite matures. RNA transcripts from the S gene are preferentially included in sporozoites that bud off from the oocyst and migrate to the salivary gland while the O gene transcripts are left within the oocyst. Although all three genes are typically eukaryotic in structure, the O gene transcript, described here, varies from the other two in core regions of the rRNA that are involved in mRNA decoding and translational termination. We now can correlate developmental progression of the parasite with changes in regions of rRNA sequence that are broadly conserved, where sequence alterations have been related to function in other systems and whose effects can be studied outside of Plasmodium. This should allow assessment of the role of translational control in parasite development.